title: Synthesis of Double-Stranded Complementary DNA from Poly(A)(+)mRNA.
abstract: The use of avian myeloblastosis virus reverse transcriptase (AMV RTase) to produce DNA copies of mRNA templates is a common and well-documented method (1-3). Briefly, the method involves synthesis of a complementary DNA strand to the mRNA from a short double-stranded region, usually provided by using an oligo(dT) primer on poly(A)(+)RNA. The enzyme does not always produce full length transcripts, but all the complementary strands are finished off with a short hairpin loop. This provides a ready-made primer for second strand synthesis, useful whether this is to be performed by more reverse transcriptase or by E. coli DNA polymerase 1 (pol 1). An idealized picture is shown in Fig. 1. Before the double-stranded cDNA (ds cDNA) copy can be cloned it is necessary to remove this hairpin loop using the single-strand specific nuclease S1. Fig. 1. Stages in the production of double-stranded cDNA from poly(A)(+)mRNA. The original RNA is represented by a solid line, while the cDNA is represented by a dashed line. Note that this diagram is not intended as an accurate representation of the enzymatic processes involved, but as a general guide to the principles of cDNA synthesis.
authors.affiliation: Inueresk Research International Limited, Musselburgh, Scotland.
authors.forename: R
authors.initials: R
authors.lastname: McGookin